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1.
Cell ; 187(19): 5316-5335.e28, 2024 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-39096902

RESUMO

Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.


Assuntos
Inflamação , Mastócitos , Camundongos Endogâmicos C57BL , Neutrófilos , Animais , Mastócitos/metabolismo , Mastócitos/imunologia , Neutrófilos/metabolismo , Neutrófilos/imunologia , Camundongos , Inflamação/metabolismo , Inflamação/imunologia , Inflamação/patologia , Leucotrieno B4/metabolismo , Transdução de Sinais , Degranulação Celular , Macrófagos/metabolismo , Macrófagos/imunologia , Armadilhas Extracelulares/metabolismo , Masculino , Feminino
2.
Immunity ; 57(10): 2296-2309.e5, 2024 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-39353440

RESUMO

The surface of the skin is continually exposed to pro-inflammatory stimuli; however, it is unclear why it is not constantly inflamed due to this exposure. Here, we showed undifferentiated keratinocytes residing in the deep epidermis could trigger a strong inflammatory response due to their high expression of pattern recognition receptors (PRRs) that detect damage or pathogens. As keratinocytes differentiated, they migrated outward toward the surface of the skin and decreased their PRR expression, which led to dampened immune responses. ZNF750, a transcription factor expressed only in differentiated keratinocytes, recruited the histone demethylase KDM1A/LSD1 to silence genes coding for PRRs (TLR3, IFIH1/MDA5, and DDX58/RIG1). Loss of ZNF750 or KDM1A in human keratinocytes or mice resulted in sustained and excessive inflammation resembling psoriatic skin, which could be restored to homeostatic conditions upon silencing of TLR3. Our findings explain how the skin's surface prevents excessive inflammation through ZNF750- and KDM1A-mediated suppression of PRRs.


Assuntos
Histona Desmetilases , Inflamação , Queratinócitos , Receptores de Reconhecimento de Padrão , Pele , Fatores de Transcrição , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Humanos , Queratinócitos/metabolismo , Animais , Camundongos , Receptores de Reconhecimento de Padrão/metabolismo , Receptores de Reconhecimento de Padrão/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Pele/imunologia , Pele/patologia , Pele/metabolismo , Inflamação/imunologia , Diferenciação Celular/imunologia , Psoríase/imunologia , Psoríase/genética , Psoríase/metabolismo , Camundongos Knockout , Inativação Gênica , Camundongos Endogâmicos C57BL , Proteínas Supressoras de Tumor
3.
Immunity ; 57(5): 1160-1176.e7, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38697118

RESUMO

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.


Assuntos
Linfócitos B , Vacinas contra Influenza , Análise de Célula Única , Humanos , Vacinas contra Influenza/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Vacinação , Anticorpos Antivirais/imunologia , Adjuvantes Imunológicos , Adjuvantes de Vacinas , Monócitos/imunologia , Polissorbatos , Esqualeno/imunologia , Imunidade Inata/imunologia
4.
Mol Cell ; 84(11): 2203-2213.e5, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38749421

RESUMO

The cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in innate immune responses to viral infection and inhibition of autoimmunity. Recent studies have suggested that micronuclei formed by genotoxic stress can activate innate immune signaling via the cGAS-STING pathway. Here, we investigated cGAS localization, activation, and downstream signaling from micronuclei induced by ionizing radiation, replication stress, and chromosome segregation errors. Although cGAS localized to ruptured micronuclei via binding to self-DNA, we failed to observe cGAS activation; cGAMP production; downstream phosphorylation of STING, TBK1, or IRF3; nuclear accumulation of IRF3; or expression of interferon-stimulated genes. Failure to activate the cGAS-STING pathway was observed across primary and immortalized cell lines, which retained the ability to activate the cGAS-STING pathway in response to dsDNA or modified vaccinia virus infection. We provide evidence that micronuclei formed by genotoxic insults contain histone-bound self-DNA, which we show is inhibitory to cGAS activation in cells.


Assuntos
Micronúcleos com Defeito Cromossômico , Nucleotidiltransferases , Transdução de Sinais , Humanos , Células HeLa , Radiação , Replicação do DNA , Dano ao DNA , Fator Regulador 3 de Interferon/metabolismo , Transcrição Gênica , Fatores Reguladores de Interferon/metabolismo , Técnicas de Inativação de Genes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Cinética , Transfecção , Nucleossomos/metabolismo , Morfolinas , Purinas , Hidroxiureia , Linhagem Celular Tumoral , Vaccinia virus/fisiologia , Vacínia/imunologia , Vacínia/metabolismo
5.
Cell ; 167(4): 1099-1110.e14, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27814507

RESUMO

As part of the Human Functional Genomics Project, which aims to understand the factors that determine the variability of immune responses, we investigated genetic variants affecting cytokine production in response to ex vivo stimulation in two independent cohorts of 500 and 200 healthy individuals. We demonstrate a strong impact of genetic heritability on cytokine production capacity after challenge with bacterial, fungal, viral, and non-microbial stimuli. In addition to 17 novel genome-wide significant cytokine QTLs (cQTLs), our study provides a comprehensive picture of the genetic variants that influence six different cytokines in whole blood, blood mononuclear cells, and macrophages. Important biological pathways that contain cytokine QTLs map to pattern recognition receptors (TLR1-6-10 cluster), cytokine and complement inhibitors, and the kallikrein system. The cytokine QTLs show enrichment for monocyte-specific enhancers, are more often located in regions under positive selection, and are significantly enriched among SNPs associated with infections and immune-mediated diseases. PAPERCLIP.


Assuntos
Citocinas/genética , Citocinas/imunologia , Infecções/imunologia , Adolescente , Adulto , Idoso , Sangue/imunologia , Feminino , Estudo de Associação Genômica Ampla , Projeto Genoma Humano , Humanos , Infecções/microbiologia , Infecções/virologia , Leucócitos Mononucleares/imunologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas
6.
Immunity ; 52(6): 1007-1021.e8, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32497523

RESUMO

N6-methyladenosine (m6A) is the most abundant RNA modification, but little is known about its role in mammalian hematopoietic development. Here, we show that conditional deletion of the m6A writer METTL3 in murine fetal liver resulted in hematopoietic failure and perinatal lethality. Loss of METTL3 and m6A activated an aberrant innate immune response, mediated by the formation of endogenous double-stranded RNAs (dsRNAs). The aberrantly formed dsRNAs were long, highly m6A modified in their native state, characterized by low folding energies, and predominantly protein coding. We identified coinciding activation of pattern recognition receptor pathways normally tasked with the detection of foreign dsRNAs. Disruption of the aberrant immune response via abrogation of downstream Mavs or Rnasel signaling partially rescued the observed hematopoietic defects in METTL3-deficient cells in vitro and in vivo. Our results suggest that m6A modification protects against endogenous dsRNA formation and a deleterious innate immune response during mammalian hematopoietic development.


Assuntos
Adenosina/química , Hematopoese/genética , Hematopoese/imunologia , Imunidade Inata/genética , RNA de Cadeia Dupla/metabolismo , Animais , Biomarcadores , Transtornos da Insuficiência da Medula Óssea/etiologia , Transtornos da Insuficiência da Medula Óssea/metabolismo , Transtornos da Insuficiência da Medula Óssea/patologia , Diferenciação Celular/genética , Modelos Animais de Doenças , Epigênese Genética , Expressão Gênica , Células-Tronco Hematopoéticas , Imunofenotipagem , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , RNA de Cadeia Dupla/química
7.
EMBO J ; 42(7): e111870, 2023 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-36178199

RESUMO

The presence of foreign nucleic acids in the cytosol is a marker of infection. Cells have sensors, also known as pattern recognition receptors (PRRs), in the cytosol that detect foreign nucleic acid and initiate an innate immune response. Recent studies have reported the condensation of multiple PRRs including PKR, NLRP6, and cGAS, with their nucleic acid activators into discrete nucleoprotein assemblies. Nucleic acid-protein condensates form due to multivalent interactions and can create high local concentrations of components. The formation of PRR-containing condensates may alter the magnitude or timing of PRR activation. In addition, unique condensates form following RNase L activation or during paracrine signaling from virally infected cells that may play roles in antiviral defense. These observations suggest that condensate formation may be a conserved mechanism that cells use to regulate activation of the innate immune response and open an avenue for further investigation into the composition and function of these condensates. Here we review the nucleic acid-protein granules that are implicated in the innate immune response, discuss general consequences of condensate formation and signal transduction, as well as what outstanding questions remain.


Assuntos
Ácidos Nucleicos , Imunidade Inata , Receptores de Reconhecimento de Padrão , Transdução de Sinais , Citosol
8.
Mol Cell ; 75(6): 1203-1217.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494035

RESUMO

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.


Assuntos
Reprogramação Celular , Endorribonucleases/metabolismo , Interferon beta/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , eIF-2 Quinase/metabolismo , Células A549 , Endorribonucleases/genética , Células HEK293 , Humanos , Interferon beta/genética , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , eIF-2 Quinase/genética
9.
Proc Natl Acad Sci U S A ; 121(43): e2409132121, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39413129

RESUMO

RNA N6-methyladenosine (m6A) demethylase AlkB homolog 5 (ALKBH5) plays a crucial role in regulating innate immunity. Lysine acylation, a widespread protein modification, influences protein function, but its impact on ALKBH5 during viral infections has not been well characterized. This study investigates the presence and regulatory mechanisms of a previously unidentified lysine acylation in ALKBH5 and its role in mediating m6A modifications to activate antiviral innate immune responses. We demonstrate that ALKBH5 undergoes lactylation, which is essential for an effective innate immune response against DNA herpesviruses, including herpes simplex virus type 1 (HSV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), and mpox virus (MPXV). This lactylation attenuates viral replication. Mechanistically, viral infections enhance ALKBH5 lactylation by increasing its interaction with acetyltransferase ESCO2 and decreasing its interaction with deacetyltransferase SIRT6. Lactylated ALKBH5 binds interferon-beta (IFN-ß) messenger RNA (mRNA), leading to demethylation of its m6A modifications and promoting IFN-ß mRNA biogenesis. Overexpression of ESCO2 or depletion of SIRT6 further enhances ALKBH5 lactylation to strengthen IFN-ß mRNA biogenesis. Our results identify a posttranslational modification of ALKBH5 and its role in regulating antiviral innate immune responses through m6A modification. The finding provides an understanding of innate immunity and offers a potential therapeutic target for HSV-1, KSHV, and MPXV infections.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase , Herpesvirus Humano 8 , Imunidade Inata , Replicação Viral , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Humanos , Replicação Viral/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Interferon beta/metabolismo , Interferon beta/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/genética , Células HEK293 , Herpesviridae/imunologia , Lipoilação
10.
RNA ; 30(10): 1292-1305, 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39009378

RESUMO

All cells in our body are equipped with receptors to recognize pathogens and trigger a rapid defense response. As a result, foreign molecules are blocked, and cells are alerted to the danger. Among the many molecules produced in response to viral infection are interferon-induced proteins with tetratricopeptide repeats (IFITs). Their role is to recognize foreign mRNA and eliminate it from the translational pool of transcripts. In the present study, we used biophysical methods to characterize the interactions between the IFIT1 protein and its partners IFIT2 and IFIT3. IFIT1 interacts with IFIT3 with nanomolar binding affinity, which did not change significantly in the presence of the preformed IFIT2/3 complex. The interactions between IFIT2 and IFIT3 and IFIT1 and IFIT2 were one order of magnitude weaker. We also present kinetic data of the interactions between the IFIT protein complex and short RNA bearing various modifications at the 5' end. We show kinetic parameters for interaction between the IFIT complex and RNA with m6Am modification. The results show that the cap-adjacent m6Am modification is a stronger signature than cap1 alone. It blocks the formation of a complex between IFIT proteins and m7Gpppm6Am-RNA much more effectively than other cap modifications. In contrast, m6A in the 5'UTR is not recognized by IFIT proteins and does not contribute to translation repression by IFIT proteins. The data obtained are important for understanding the regulation of expression of genetic information. They indicate that 2'-O and m6Am modifications modulate the availability of mRNA molecules for proteins of innate immune response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ligação Proteica , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Cinética , Proteínas/metabolismo , Proteínas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética
11.
Annu Rev Microbiol ; 75: 19-47, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33492978

RESUMO

In less than two decades, three deadly zoonotic coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have emerged in humans, causing SARS, MERS, and coronavirus disease 2019 (COVID-19), respectively. The current COVID-19 pandemic poses an unprecedented crisis in health care and social and economic development. It reinforces the cruel fact that CoVs are constantly evolving, possessing the genetic malleability to become highly pathogenic in humans. In this review, we start with an overview of CoV diseases and the molecular virology of CoVs, focusing on similarities and differences between SARS-CoV-2 and its highly pathogenic as well as low-pathogenic counterparts. We then discuss mechanisms underlying pathogenesis and virus-host interactions of SARS-CoV-2 and other CoVs, emphasizing the host immune response. Finally, we summarize strategies adopted for the prevention and treatment of CoV diseases and discuss approaches to develop effective antivirals and vaccines.


Assuntos
COVID-19/virologia , Infecções por Coronavirus/virologia , Coronavirus/fisiologia , SARS-CoV-2/fisiologia , Animais , COVID-19/imunologia , COVID-19/transmissão , Coronavirus/classificação , Coronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/transmissão , Interações Hospedeiro-Patógeno , Humanos , SARS-CoV-2/genética , Tratamento Farmacológico da COVID-19
12.
J Biol Chem ; 300(5): 107233, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38552738

RESUMO

The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.


Assuntos
Inflamassomos , Proteínas Mitocondriais , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas de Transporte de Fosfato , Animais , Humanos , Camundongos , Células HEK293 , Inflamassomos/metabolismo , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Transporte de Fosfato/genética , Ubiquitinação , Linhagem Celular , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Técnicas de Silenciamento de Genes
13.
Am J Hum Genet ; 109(12): 2126-2140, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459979

RESUMO

Chromosome gains are detrimental for the development of the human embryo. As such, autosomal trisomies almost always result in spontaneous abortion, and the rare embryos surviving until live birth suffer from a plethora of pathological defects. There is no treatment currently available to ameliorate the consequences of trisomies, such as Down syndrome (trisomy of chromosome 21). Identifying the source of the phenotypes observed in cells with extra chromosomes is crucial for understanding the underlying molecular causes of trisomy syndromes. Although increased expression of the genes localized on the extra chromosome triggers several pathological phenotypes, an alternative model suggests that global, aneuploidy-associated changes in cellular physiology also contribute to the pathology. Here, we compare the molecular consequences of trisomy syndromes in vivo against engineered cell lines carrying various chromosome gains in vitro. We point out several phenotypes that are shared by variable trisomies and, therefore, might be caused by the presence of an extra chromosome per se, independent of its identity. This alternative view may provide useful insights for understanding Down syndrome pathology and open additional opportunities for diagnostics and treatments.


Assuntos
Síndrome de Down , Trissomia , Feminino , Gravidez , Humanos , Trissomia/genética , Síndrome de Down/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 21 , Aneuploidia
14.
RNA ; 29(3): 273-281, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36596670

RESUMO

Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the immune response. Here, we review how Z-RNA features present in cellular RNAs-especially containing retroelements-could be recognized by a family of winged helix proteins, with an impact on host defense. We also discuss how mutations to specific Z-contacting amino acids disrupt their ability to stabilize Z-RNA, resulting in functional losses. We end by highlighting knowledge gaps in the field, which, if addressed, would significantly advance this active area of research.


Assuntos
DNA Forma Z , RNA , RNA/química , Adenosina Desaminase/metabolismo , Imunidade Inata/genética , Aminoácidos , Biologia
15.
J Virol ; 98(10): e0105524, 2024 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-39212382

RESUMO

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects of a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated, and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection. IMPORTANCE: Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that co-opts cellular mechanisms to support viral processes that can reprogram the host transcriptional profile. Such viral-directed transcriptional changes and the pro- or anti-viral outcomes remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV-infected mammalian cells, along with other cellular and immune response genes. We now define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. We show that during ZIKV infection, the integrated stress response pathway stimulates ATF3 which enhances the innate immune response to antagonize ZIKV infection. This study establishes a link between viral-induced stress response and transcriptional regulation of host defense pathways and thus expands our knowledge of virus-mediated transcriptional mechanisms and transcriptional control of interferon-stimulated genes during ZIKV infection.


Assuntos
Fator 3 Ativador da Transcrição , Interações Hospedeiro-Patógeno , Imunidade Inata , Infecção por Zika virus , Zika virus , Fator 3 Ativador da Transcrição/metabolismo , Fator 3 Ativador da Transcrição/genética , Zika virus/imunologia , Humanos , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia , Células A549 , Interações Hospedeiro-Patógeno/imunologia , Fator 4 Ativador da Transcrição/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Replicação Viral , Estresse Fisiológico , Células Vero , Chlorocebus aethiops
16.
J Virol ; 98(7): e0058424, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38888344

RESUMO

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has resulted in substantial morbidity and mortality. The basis of severe disease in humans is difficult to determine without the use of experimental animal models. Mice are resistant to infection with ancestral strains of SARS-CoV-2, although many variants that arose later in the pandemic were able to directly infect mice. In almost all cases, viruses that naturally infected mice or were engineered to enable mouse infection required mouse passage to become virulent. In most cases, changes in structural and nonstructural changes occurred during mouse adaptation. However, the mechanism of increased virulence in mice is not understood. Here, using a recently described strain of mouse-adapted SARS-CoV-2 (rSARS2-MA30N501Y), we engineered a series of recombinant viruses that expressed a subset of the mutations present in rSARS2-MA30N501Y. Mutations were detected in the spike protein and in three nonstructural proteins (nsp4, nsp8, and nsp9). We found that infection of mice with recombinant SARS-CoV-2 expressing only the S protein mutations caused very mild infection. Addition of the mutations in nsp4 and nsp8 was required for complete virulence. Of note, all these recombinant viruses replicated equivalently in cultured cells. The innate immune response was delayed after infection with virulent compared to attenuated viruses. Further, using a lineage tracking system, we found that attenuated virus was highly inhibited in the ability to infect the parenchyma, but not the airway after infection. Together, these results indicate that mutations in both the S protein and nonstructural proteins are required for maximal virulence during mouse adaptation.IMPORTANCEUnderstanding the pathogenesis of coronavirus disease 2019 (COVID-19) requires the study of experimental animals after infection with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). For this purpose, several mouse-adapted SARS-CoV-2 strains have been developed. Here, using a strain of mouse-adapted virus that causes a range of diseases ranging from mild to severe, we show that mutations in both a structural protein [spike (S) protein] and nonstructural proteins are required for maximal virulence. Thus, changes in the S protein, the most widely studied viral protein, while required for mouse adaptation, are not sufficient to result in a virulent virus.


Assuntos
COVID-19 , Modelos Animais de Doenças , Mutação , SARS-CoV-2 , Proteínas não Estruturais Virais , Animais , Camundongos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , COVID-19/virologia , SARS-CoV-2/patogenicidade , SARS-CoV-2/genética , Virulência , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Chlorocebus aethiops , Replicação Viral , Feminino
17.
J Virol ; 98(7): e0068624, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38888343

RESUMO

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.


Assuntos
Proteínas do Capsídeo , Doenças dos Peixes , Evasão da Resposta Imune , Imunidade Inata , Nodaviridae , Nucleotidiltransferases , Infecções por Vírus de RNA , Transdução de Sinais , Replicação Viral , Animais , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/metabolismo , Interferons/metabolismo , Interferons/imunologia , Bass/imunologia , Bass/virologia , Bass/metabolismo , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia
18.
J Virol ; 98(1): e0183023, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38088560

RESUMO

Usutu virus (USUV) and West Nile virus (WNV) are closely related emerging arboviruses belonging to the Flavivirus genus and posing global public health concerns. Although human infection by these viruses is mainly asymptomatic, both have been associated with neurological disorders such as encephalitis and meningoencephalitis. Since USUV and WNV are transmitted through the bite of an infected mosquito, the skin represents the initial site of virus inoculation and provides the first line of host defense. Although some data on the early stages of WNV skin infection are available, very little is known about USUV. Herein, USUV-skin resident cell interactions were characterized. Using primary human keratinocytes and fibroblasts, an early replication of USUV during the first 24 hours was shown in both skin cells. In human skin explants, a high viral tropism for keratinocytes was observed. USUV infection of these models induced type I and III interferon responses associated with upregulated expression of various interferon-stimulated genes as well as pro-inflammatory cytokine and chemokine genes. Among the four USUV lineages studied, the Europe 2 strain replicated more efficiently in skin cells and induced a higher innate immune response. In vivo, USUV and WNV disseminated quickly from the inoculation site to distal cutaneous tissues. In addition, viral replication and persistence in skin cells were associated with an antiviral response. Taken together, these results provide a better understanding of the pathophysiology of the early steps of USUV infection and suggest that the skin constitutes a major amplifying organ for USUV and WNV infection.IMPORTANCEUsutu virus (USUV) and West Nile virus (WNV) are closely related emerging Flaviviruses transmitted through the bite of an infected mosquito. Since they are directly inoculated within the upper skin layers, the interactions between the virus and skin cells are critical in the pathophysiology of USUV and WNV infection. Here, during the early steps of infection, we showed that USUV can efficiently infect two human resident skin cell types at the inoculation site: the epidermal keratinocytes and the dermal fibroblasts, leading to the induction of an antiviral innate immune response. Moreover, following cutaneous inoculation, we demonstrated that both viruses can rapidly spread, replicate, and persist in all distal cutaneous tissues in mice, a phenomenon associated with a generalized skin inflammatory response. These results highlight the key amplifying and immunological role of the skin during USUV and WNV infection.


Assuntos
Infecções por Flavivirus , Flavivirus , Tropismo Viral , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Humanos , Camundongos , Antivirais , Culicidae , Infecções por Flavivirus/virologia , Interferons , Febre do Nilo Ocidental/virologia , Pele/imunologia , Pele/patologia , Pele/virologia , Técnicas In Vitro
19.
J Virol ; : e0123224, 2024 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-39431848

RESUMO

During virus replication in cultured cells, copy-back defective viral genomes (cbDVGs) can arise. CbDVGs are powerful inducers of innate immune responses in vitro, but their occurrence and impact on natural infections of human hosts remain poorly defined. We asked whether cbDVGs were generated in the brain of a patient who succumbed to subacute sclerosing panencephalitis (SSPE) about 20 years after acute measles virus (MeV) infection. Previous analyses of 13 brain specimens of this patient indicated that a collective infectious unit (CIU) drove lethal MeV spread. In this study, we identified 276 replication-competent cbDVG species, each present in over 100 copies in the brain. Six species were detected in multiple forebrain locations, implying that they travelled long-distance with the CIU. The cbDVG to full-length genomes ratio was often close to 1 (0.6-1.74). Most cbDVGs were 324-2,000 bases in length, corresponding to 2%-12% of the full-length genome; all are predicted to have complementary terminal sequences. If improperly encapsidated, these sequences have the potential to form double-stranded structures that can induce innate immune responses. To assess this, we examined the transcriptome of all brain specimens. Several interferon and inflammatory response genes were upregulated, but upregulation levels did not correlate with cbDVG levels in the specimens. Thus, the CIU that drove MeV pathogenesis in this brain includes, in addition to two complementary full-length genome populations, many locally restricted and few widespread cbDVG species. The widespread cbDVG species may have been positively selected but how they impacted pathogenesis remains to be determined.IMPORTANCECopy-back defective viral genomes (cbDVGs) can drive virus-host interactions. They can suppress virus replication directly, by competing with full-length genomes, or indirectly by stimulating antiviral immunity. In vitro, cbDVG can slow down infections and promote persistence, but there is limited documentation of their presence in human hosts or of their impact on disease. We had the unique opportunity to analyze the brain of a patient who succumbed to subacute sclerosing panencephalitis, a rare but lethal consequence of measles. We detected more than 270 distinct cbDVG species; most were restricted to one specimen, but several reached all lobes of the forebrain, suggesting positive selection. Our analyses provide the missing knowledge of the diversity of cbDVG in a natural infection of a human host. They also reveal that a collective infectious unit that caused lethal human brain disease includes few widespread cbDVG, in addition to two ubiquitous complementary full-length genome populations.

20.
FASEB J ; 38(8): e23628, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38661032

RESUMO

Cancer cells frequently exhibit hyperactivation of transcription, which can lead to increased sensitivity to compounds targeting the transcriptional kinases, in particular CDK9. However, mechanistic details of CDK9 inhibition-induced cancer cell-selective anti-proliferative effects remain largely unknown. Here, we discover that CDK9 inhibition activates the innate immune response through viral mimicry in cancer cells. In MYC over-expressing prostate cancer cells, CDK9 inhibition leads to the gross accumulation of mis-spliced RNA. Double-stranded RNA (dsRNA)-activated kinase can recognize these mis-spliced RNAs, and we show that the activity of this kinase is required for the CDK9 inhibitor-induced anti-proliferative effects. Using time-resolved transcriptional profiling (SLAM-seq), targeted proteomics, and ChIP-seq, we show that, similar to viral infection, CDK9 inhibition significantly suppresses transcription of most genes but allows selective transcription and translation of cytokines related to the innate immune response. In particular, CDK9 inhibition activates NFκB-driven cytokine signaling at the transcriptional and secretome levels. The transcriptional signature induced by CDK9 inhibition identifies prostate cancers with a high level of genome instability. We propose that it is possible to induce similar effects in patients using CDK9 inhibition, which, we show, causes DNA damage in vitro. In the future, it is important to establish whether CDK9 inhibitors can potentiate the effects of immunotherapy against late-stage prostate cancer, a currently lethal disease.


Assuntos
Quinase 9 Dependente de Ciclina , Imunidade Inata , Humanos , Masculino , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo
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